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Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellanii trophozoites and cysts
Author(s) -
Shi Lei,
Stachon Tanja,
Latta Lorenz,
Ibrahem Elhawy Mohamed,
Orosz Erika,
Kiderlen Albrecht F.,
Seitz Berthold,
Bischoff Marukus,
Szentmáry Nóra
Publication year - 2019
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2019.5059
Subject(s) - calcein , trypan blue , microbiology and biotechnology , staining , agar , in vitro , biology , chemistry , bacteria , biochemistry , genetics , membrane
Purpose To compare LDH release assay, trypan blue and fluorescent stainings and non‐nutrient Escherichia coli (E. coli) plate assay in determining treatment efficacy of antiamoebic‐agents against Acanthamoeba castellanii trophozoites and cysts, in‐vitro. Methods A. castellanii strain 1BU‐trophozoites/cysts were challenged with the antiamoebic‐agents 0.02% polyhexamethylen‐biguanid (PHMB), 0.1% propamidin‐isethionate (PD) and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH‐release and trypan blue assays. The fluorescent dyes Hoechst 33343, calcein‐AM, and ethidium‐homodimer‐1 were tested for their abilities to differentiate between viable and dead 1BU‐trophozoites/cysts following treatment, a non‐nutrient agar E. coli plate assay was applied to monitor the outgrowth of 1BU‐trophozoites/cysts challenged with antiamoebic‐agents. Results All three antiamoebic‐agents induced a significant LDH‐release from trophozoites, when compared to controls (p < 0.0001). There was a negligible trypan blue, Hoechst33342, calcein‐AM and ethidium‐homodimer‐1 staining in untreated 1BU‐throphozoites and cysts. Percentage of trypan blue positive trophozoites/cysts was 59.3 ± 1.5% /100% after challenge with PHMB, 100%/99.3 ± 0.5% upon PD treatment, and 95.6 ± 2.5%/77.6 ± 5.1% when using MF. Hoechst 33342 stained 100%/100%, ethidium‐homodimer‐1 100%/100%, calcein‐AM 75.33 ± 4.04%/60.67 ± 3.51% of PHMB treated trophozoites/cysts. Hoechst 33342 and ethidium‐homodimer‐1 were 100%/100%, calcein‐AM 9.67 ± 2.52%/36.67 ± 4.51% positive in PD‐treated trophozoites/cysts. MF treatment resulted in 100%/100% Hoechst 33342 and ethidium‐homodimer‐1 positive trophozoites/cysts, while none of them became positive for calcein‐AM. On non‐nutrient agar E. coli plates, in controls and MF treated 1BU trophozoites/cysts, new trophozoites appeared within 24 hr, and encystment occured after 5 weeks of cultivation. In PHMB and PD treated 1BU‐throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 hr, which failed to form new cysts within 5 weeks. Conclusions None of the enzymatic‐ and dye‐based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non‐nutrient agar E. coli plate assay, suggesting that the culture‐based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.